NMDA receptors in the lateral preoptic hypothalamus are essential for sustaining NREM and REM sleep

The lateral preoptic (LPO) hypothalamus is a center for NREM and REM sleep induction and NREM sleep homeostasis. Although LPO is needed for NREM sleep, we found that calcium signals were, surprisingly, highest in REM sleep. Furthermore, and equally surprising, NMDA receptors in LPO were the main drivers of excitation. Deleting the NMDA receptor GluN1 subunit from LPO abolished calcium signals in all cells and produced insomnia. Mice of both sexes had highly fragmented NREM sleep-wake patterns and could not generate conventionally classified REM sleep. The sleep phenotype produced by deleting NMDA receptors depended on where in the hypothalamus the receptors were deleted. Deleting receptors from the anterior hypothalamic area did not influence sleep-wake states. The sleep fragmentation originated from NMDA receptors on GABA neurons in LPO. Sleep fragmentation could be transiently overcome with sleeping medication (zolpidem) or sedatives (dexmedetomidine). By contrast, fragmentation persisted under high sleep pressure produced by sleep deprivation - mice had a high propensity to sleep but woke up. By analyzing changes in delta power, sleep homeostasis (also referred to as “sleep drive”) remained intact after NMDA receptor ablation. We suggest NMDA glutamate receptor activation stabilizes firing of sleep-on neurons, and that mechanisms of sleep maintenance differ from that of the sleep drive itself.


Introduction
Both NREM and REM sleep are partly controlled by the preoptic (PO) hypothalamus (Nauta, 1946;McGinty and Sterman, 1968;Sherin et al., 1996;John and Kumar, 1998;Lu et al., 2000;Lu et al., 2002;Szymusiak et al., 2007). In this region, GABA/peptidergic neurons, e.g. GABA/galanin neurons, contribute to NREM sleep induction and sleep homeostasis (Sherin et al., 1996;Zhang et al., 2015;Chung et al., 2017;Kroeger et al., 2018;Ma et al., 2019;Reichert et al., 2019). To stay asleep, it seems reasonable to assume that these sleep-promoting neurons would have to stay "on". Indeed, lesioning of lateral PO (LPO) neurons in rats reduces the amounts of NREM or REM sleep, depending on the location of the lesion (Lu et al., 2000). But molecular factors that keep LPO sleep-promoting neurons firing and so govern the lengths of NREM and REM sleep episodes are not known.
One critical factor maintaining sleep could be NMDA-type glutamate receptors. These channels, especially when located extrasynaptically, can provide tonic excitation (Sah et al., 1989;Papouin et al., 2012;Neupane et al., 2021). Indeed, NMDA receptor activation promotes sleep. In the fruit fly Drosophila, genetic knockdown of NMDA receptors in brain reduces total sleep time (Tomita et al., 2015). In rodents, NMDA receptor antagonists reduce and agonists enhance NREM sleep (Tatsuki et al., 2016;Burgdorf et al., 2019). Furthermore, patients with autoimmunity to the essential GluN1 subunit of NMDA receptors often suffer insomnia (Dalmau et al., 2019;Arino et al., 2020). Because NMDA receptors are expressed throughout the brain (Moriyoshi et al., 1991;Laurie and Seeburg, 1994;Monyer et al., 1994), these effects on sleep could come from interference with many circuits.
Calcium entry through NMDA receptors has also been suggested to be part of the sleep homeostasis mechanism that tracks time spent awake (Liu et al., 2016). Even if sleep is poor, wakefulness still cannot be sustained beyond a certain limit. This limit is thought to be imposed by the process of sleep homeostasis, the increasing drive to enter NREM sleep as wakefulness continues (Borbely et al., 2016). Sleep homeostasis is operationally studied as an increase in NREM delta power after sleep deprivation (Hanlon et al., 2011;Franken, 2013;Greene et al., 2017;Deboer, 2018). Given that the PO hypothalamus is one of the key regions controlling sleep homeostasis Donlea et al., 2017;Ma et al., 2019;Reichert et al., 2019), we were keen to test how NMDA receptors in the LPO area influence this process.
We found that the whole LPO area has selectively raised calcium activity in REM sleep, and this calcium entry depends on NMDA receptors. In this study we deleted the GluN1 NMDA receptor subunit in the LPO hypothalamus and obtained a marked "insomnia" phenotype with high NREM sleep-wake fragmentation and greatly diminished REM sleep. Sleep homeostasis, however, was unaffected by removing NMDA receptors from LPO. The NREM sleep-wake fragmentation effect is selective for GluN1 expression in GABA neurons.
All mice were housed at a maximum of five mice per cage with food and water ad libitum and maintained under the same conditions (21±1 °C, reversed 12/12h dark/light cycle). Zeitgeber time (ZT) 0 is defined as the time when the light was switched on (16:00) and ZT12 is defined as the time of light off (4:00). For behavioral experiments, mice were singly housed, and experiments performed during dark phase (ZT12-24) unless otherwise specified, while photometry recordings were performed during light phase (ZT0-12).

Surgeries
All surgeries used adult male and female mice, 8-12 weeks old and were performed under deep general anesthesia with isoflurane (3% induction/ 2% maintenance) and under sterile conditions. Before starting the surgery, mice were injected subcutaneously (s.c.) with Buprenorphine (Vetergesic 0.3 mg/mL, 1:20 dilution in 0.9% sterile saline solution, final 0.1mg/kg) and Carprofen (Rimadyl 50mg/mL, 1:50 dilution in 0.9% sterile saline solution, final 5 mg/kg) and then placed in a stereotaxic frame. Mouse core temperature was constantly checked by rectal probe while respiration rate was regularly checked by eye.
For AAV injections, the virus was injected at a rate of 0.1 μL/min using Hamilton microliter #701 10 μL syringes and a stainless-steel needle (33-gauge, 15 mm long). LPO coordinates used for bilateral injection sites were relative to Bregma: AP:+0.40 mm, ML: -/+ 0.75 mm, DV was consecutive, injecting half volume at +5.20 mm and half at +5.15 mm. A total volume of 0.3 μL each side was injected. Control AHA coordinates used for bilateral injections sites were relative to Bregma: AP: -0.58 mm, ML: -/+ 0.65 mm, DV: +5.60 mm and +5.50 mm for consecutive injections.
For all surgeries, the wound was sewed around the head stage and the mouse was left recovering in a heat box. All instrumented mice were single housed to avoid lesions to the head stage. After surgery, mice injected with AAVs were allowed 1 month for recovering and for the viral transgenes to adequately express before being fitted with Neurologger 2A devices (see below) and undergoing any experimental procedures.

EEG/EMG recordings and analysis
EEG and EMG traces were recorded using Neurologger 2A devices as described previously (Vyssotski et al., 2009;Anisimov et al., 2014;Gelegen et al., 2014), at a sampling rate of 200 Hz. The data obtained from the Neurologger 2A were downloaded and visualized using Spike2 Software (Cambridge Electronic Design, Cambridge, UK). The EEG was high pass filtered (0.5Hz, -3dB) using a digital filter, while EMG was band pass filtered between 5-45 Hz (-3dB). To define the vigilance states of Wake,NREM and REM sleep, Hz) and theta (5-10 Hz) power were calculated, as well as theta:delta power ratio and the EMG integral. Automated sleep scoring was performed using a Spike2 script and the result was manually corrected. For the three vigilance states, percentage amounts were calculate using costume Spike2 scripts. For sleep architecture analysis, costume MATLAB scripts were used. For stage transitions, we calculated each stage change reported in the hypnogram. The baseline number of transitions for animals lacking NMDA receptors has been represented as percentages over the control group. For sleep-deprived mice, we represented the transition numbers after 6hr SD as a percentage over the baseline value for each animal, in both control and experimental groups. Fast Fourier transformation (512 points) was used to calculate EEG power spectra. oxygenation and transferred to oxygenated standard aCSF (in mM: NaCl 120, KCl 3.5, NaH 2 PO 4 1.25, NaHCO 3 25, glucose 10, MgCl 2 1, CaCl 2 2) solution for at least 1 hour at room temperature. Slices were transferred to a submersion recording chamber and were continuously perfused at a rate of 4-5ml/min with fully oxygenated aCSF at room temperature. For whole-cell recording, patch pipettes at 4-6 MΩ were pulled from borosilicate glass capillaries (1.5mm OD, 0.86 mm ID, Harvard Apparatus, #GC150F-10) and filled with intracellular solution containing in mM: 128 CsCH 3 SO 3 , 2.8 NaCl, 20 HEPES, 0.4 EGTA, 5 TEA-Cl, 2 Mg-ATP, 0.5 NaGTP (pH 7.35, osmolality 285 mOsm). 0.1% Neurobiotin was included in the intracellular solutions to identify the cell position and morphology following recording. Recordings were performed using a Multiclamp 700B amplifier (Molecular Devices. CA). Access and input resistances were monitored throughout the experiments. The access resistance was typically < 20 MΩ, and results we discarded if resistance changed by more than 20%.
GFP-positive neurons were visually identified and randomly selected. For AMPA and NMDA currents, a bipolar stimulus microelectrode (MX21AEW, FHC) was placed 100-200 μm away caudally from the recording site. The intensity of stimulus (10 ms) was adjusted to evoke a measurable, evoked EPSC in recording cells. AMPA and NMDA mixed currents were measured at a holding potential of +40 mV. After obtaining at least 10 sweeps of stable mixed currents, D-AP5 (50 μM) was perfused in the bath solution for 15 min and AMPA currents were measured. NMDA currents were obtained by subtracting AMPA currents from mixed currents off-line. The peak amplitude of both currents was used for AMDA/NMDA ratio analysis. For sEPSCs, GFP-positive LPO neurons were voltage clamped at -70 mV. A stable baseline recording was obtained for 5-10 min. Frequency, amplitude, rise & decay time constants of sEPSCs were analyzed off-line with the Mini Analysis (Synaptosoft). Frequency was obtained from 2 min of recording. All recordings were made in the presence of picrotoxin (100 μM).
For immunohistochemistry following electrophysiological recordings, brain slices were post-fixed in 4% PFA overnight at 4 °C. PFA was then washed away 3 times for 10 min in 1x PBS and slices were blocked and permeabilized in 20% NGS or 2% Bovine Serum Albumin (BSA) for 3 hours shaking. Primary anti-GFP antibody to trace viral distribution was diluted in 2% NGS, 0.5/0.7% TritonX in PBS overnight at 4 °C shaking. After 4 washes in 1x PBS for 10min each, secondary antibody was diluted in 2% NGS and 0.5% TritonX for 3 hours at RT and shaking. After washes and to track Neurobiotin-filled neurons recorded by electrophysiology, an Alexa594-conjugated streptavidin (Invitrogen) was diluted 1:500 in 1% NGS, 0.5% TritonX and slices were incubated for 2-3 hours at RT. Four washes of 15 min and subsequent DAPI incubation for 10 min were performed before slices were mounted on glass slides.

Calcium Photometry
Following 4 weeks of recovery, mice were acclimatized to the testing environment for at least 2 hours before behavioral experiments and then recorded for 6 hours during the light period. The light source was a 473-nm diode-pumped solid state (DPSS) laser with fiber coupler (Shanghai Laser & Optics century Co.) and adjustable power supply (Shanghai Laser & Optics century Co.), controlled by a Grass SD9 stimulator. A lock-in amplifier (SR810, Stanford Research Systems, California, USA) was used to drive the laser at 125 Hz TTL pulses with an average power of 80 μW at the tip of the fiber directly connected to the mouse. The light source was coupled to a fluorescence cube (FMC_GFP_FC, Doric Lenses) through an optical fiber patch cord (Ø 200 μm,0.22 NA,Doric Lenses). From the filter cube, an optical patch cord (Ø 200 μm, 0.37 NA, Doric Lenses) was connected to the monofiber chronically implanted in the mouse brain using ceramic sleeves (Thorlabs). The GCaMP6s output was then filtered at 500-550 nm through the fluorescence cube, converted to a voltage by a photodiode (APD-FC, Doric Lenses) and then amplified by the lock-in amplifier with a time constant of 30 ms. Finally, the signal was digitalized using a CED 1401 Micro box (Cambridge Electronic Design, Cambridge, UK) and recorded at 200 Hz using Spike2 software (Cambridge Electronic Design, Cambridge, UK). Photometry, EEG and EMG data were aligned offline using Spike2 software and analyzed using custom MATLAB (MathWorks) scripts. For each experiment, the photometry signal F was normalized to baseline using the function ΔF/F = (F-F 0 )/F 0 , where F 0 is the mean fluorescence across the signal analyzed. When scatter plots of ΔF/F levels were plotted for each behavioral state, ΔF/F values were obtained by calculating the average F over a 50s rolling window, with a 0.2 Hz sampling rate over 70-90 minutes of photometry recordings. We applied a custom Matlab script to correct the baseline photometry values for photobleaching and photometry signal drift during long recordings.
The three sequences were referred to as shRNA-GluN1-1.1, -2 or -3 (the underlined sequences are the 22-mers specific for the GluN1 subunit): shRNA-GluN1.1 targeted the GluN1 sequence at 1800bp (600aa) of the coding sequence: 3'-TGCTGTTGACAGTGAGCGAACTGACCCTGTCCTCTGCCATTAGTGAAGCC ACAGATGTAATGGCAGAGGACAGGGTCAGTGTGCCTACTGCCTCGGA-5' shRNA-GluN1.2 targeted the GluN1 sequence from 2565bp (855aa) of the coding sequence: 3'-TGCTGTTGACAGTGAGCGCGCCGTGAACGTGTGGAGGAAGTAGTGAAGC CACAGATGTACTTCCTCCACACGTTCACGGCTTGCCTACTGCCTCGGA-5' shRNA-GluN1.3 targeted the GluN1 sequence at 2215bp (738aa) of the coding region: 3'-TGCTGTTGACAGTGAGCGCGGAGTTTGAGGCTTCACAGAATAGTGAAGC CACAGATGTATTCTGTGAAGCCTCAAACTCCATGCCTACTGCCTCGGA-5' As a control for shRNA-GluN1 sequences, an shRNA scramble hairpin was used, not complementary to sequences in the mouse genome. The shRNA-scramble (scr) sequence was: The three shRNA-GluN1 hairpins and the shRNA-scr hairpin were cloned into the pPRIME vector to be then expressed and tested in HEK293 cells. To establish shRNA efficiencies in knocking down the NMDA GluN1 subunit expression, a plasmid was constructed expressing GluN1-2A-mCherry under the control of the CMV promoter. Each GluN1-shRNA pPRIME plasmid was then transfected into HEK293 cells together with pGluN1-2A-mCherry. After 60 hours in culture, mCherry fluorescence was quantified. The GluN1.3 shRNA produced lower fluorescence intensity, and thus higher inhibition of GluN1 expression, and it was therefore cloned into an AAV transgene cassette in an inverse orientation flanked by lox sites, as we described previously , to produce pAAV-flex-GFP-shRNA-GluN1 with an hdc promoter fragment (this plasmid has been deposited at Addgene, number 182502) which was then packaged into AAV capsids (as above). The AAV transgene expresses GFP as well as shRNA-GluN1. For the controls, we packaged pAAV-flex-GFP-shRNA-scramble (this plasmid has been deposited at Addgene, number 182503).

Experimental Design and Statistical Analysis
Origin, MATLAB and GraphPad Prism 8 were used for graphs and statistical analysis. Data collection and experimental procedure conditions were randomized. The experimenter was not blinded during the procedures. Data are presented as mean ± standard error of the means (SEM). The normality of each data set distribution was tested using the Kolmogorov-Smirnoff test. Paired/Unpaired two-tailed Student's t test or one-way ANOVA were used to compare groups when only one variable was present. For longitudinal measurements, or measurements with two separate independent variables, a two-way repeated measures ANOVA, or a simple two-way ANOVA followed by post-hoc Sidak, Tukey, or Dunnett tests were performed. F and p values reported in the text are relative to the ANOVA statistical tests. Significantly different time points obtained through a multiple comparison analysis in the context of a two-way ANOVA test are indicated in the Figures, where *p < 0.05, ** p < 0.005, *** p < 0.0005, † p < 0.00005. When the data were not normally distributed, a Mann-Whitney test was performed. Statistical significance was considered when *p < 0.05, ** p < 0.005, *** p < 0.0005.

Activity in LPO neurons is highest during REM sleep
We first recorded neuronal activity in all LPO neurons using photometry with the calcium sensor GCaMP6s. Mice were injected in LPO with AAV-GCaMP6s ( Fig. 1A-B). The highest calcium activity occurred during REM sleep episodes, especially at the beginning and end of the episodes ( Figure 1C-D). During NREM sleep, LPO neurons showed a more sporadic and spiky activity, and during wakefulness only low activity. By plotting scaled means of the GCaMP6s signal against EMG signal (
We examined if NMDA-receptor currents were deleted from ΔGluN1-LPO neurons compared with control GFP-LPO mice injected with AAV-GFP by recording evoked excitatory post-synaptic currents (eEPSCs) from ex-vivo acute slices prepared from the PO area ( Fig. 2D-E). Both NMDA receptor-mediated slow currents (hundreds of milliseconds) and AMPA receptor-mediated fast currents (few milliseconds) were found on LPO neurons of GFP-LPO mice. NMDA receptor-mediated currents were smaller and the NMDA/AMPA ratio was significantly reduced in cells from ΔGluN1-LPO mice (two-tailed Mann Whitney test, U=10, p = 0.02. ΔGluN1-LPO (n = 8); n= 5, Fig 2E).
We next tested how NMDA receptor deletion from LPO neurons influenced intracellular calcium transitions. We co-injected into the LPO area of Grin1 lox mice AAV-iCre-2A-mCherry and AAV-flex-GCaMP6s, so that only neurons expressing Cre recombinase
In addition to sleep loss and reduced cortical theta power, ΔGluN1-LPO mice had a highly fragmented sleep-wake phenotype: they lacked long Wake and NREM sleep episodes, as they had significantly more Wake Fig. 4I).

Hypothalamic region-specific effect of NMDA receptor ablation on sleep-wake fragmentation
As a control, we tested if deleting NMDA receptors in a region neighboring the PO area, the anterior hypothalamic area (AHA), caused sleep loss or fragmentation. Bilateral injection of AAV-iCre-2A-Venus and AAV-GFP into the AHA of Grin1 lox mice, to generate ΔGluN1-AHA and GFP-AHA mice respectively (Fig. 5A 5D). Therefore, the fragmented sleep phenotype produced by deleting NMDA receptors originates region-selectively in the preoptic hypothalamus.

The insomnia of mice with NMDA receptors deleted from the LPO hypothalamus persists under high sleep pressure
In both fruit flies and mice, calcium entry through NMDA-type ionotropic glutamate-gated receptors has been proposed to signal the sleep homeostatic process (Liu et al., 2016;Tatsuki et al., 2016;Raccuglia et al., 2019). To investigate if the fragmented sleep of ΔGluN1-LPO mice persisted under high sleep pressure and if NMDA receptors in LPO were required for sleep homeostasis, we performed 6h of sleep deprivation (SD) at the onset of the "lights on" period (ZT0) when the sleep drive is highest (Fig. 6A). Although ΔGluN1-LPO mice were awake and moving during the sleep deprivation, there were several indications that they were under high sleep pressure. During the sleep deprivation, the EEG theta power in ΔGluN1-LPO mice was greatly reduced (red trace in Fig showing that, by this measure, sleep homeostasis was intact. The typical diurnal variation in EEG delta power over 24 hours seen in control mice was also still present in ΔGluN1-LPO mice ( Fig. 6D and E right panels). However, ΔGluN1-LPO mice were incapable of recuperating the sleep lost during sleep deprivation (Fig. 6F Fig. 6H, right panel). The persistence of fragmentation after SD in ΔGluN1-LPO mice is noteworthy, as under increased sleep pressure, quantified by the delta power rebound, sleep is deeper compared with baseline levels. These data suggest that ΔGluN1-LPO mice were sleepy but could not stay asleep.

Sedatives and sleeping medication transiently improve sleep of mice with NMDA receptors deleted from the LPO hypothalamus
We investigated whether drugs that induce NREM-like sleep, dexmedetomidine (Dex) which is used in intensive care units for long-term sedation (Adams et al., 2013), and zolpidem (Ambien), a widely prescribed sleeping medication ( Fig. 7F, right panel).

Sleep fragmentation but not sleep loss is produced by selective NMDA GluN1 subunit knock-down in GABA LPO neurons but not glutamate LPO neurons
To investigate the NR1-expressing LPO cell types involved in regulating sleep, we could not use Cre recombinase to ablate the Grin1 gene because no cell type-selective promoters are available to restrict NR1 deletion selectively to subtypes of LPO cells without also affecting other brain areas. We therefore decided to use shRNA transgenes to reduce GluN1 expression cell-type selectively, for example, in GABAergic or glutamatergic cells in LPO (see Materials & Methods). First, we tested the efficacy of three different shRNAs to knockdown recombinant GluN1-mCherry cDNA expression ( Fig. 8A and B). Having identified a suitable shRNA, shRNA3, that significantly reduced GluN1-mCherry cDNA expression compared with scrambled shRNA (one-way ANOVA: F (3, 44) = 9.13, p < 0.0001; Tukey's post-hoc test: Scramble vs. shRNA3 p = 0.0007, shRNA1 vs. shRNA3 p = 0.0003, shRNA2 vs. shRNA3 p = 0.0008, n = 10 transfections, , Fig. 8B), the shRNA was cloned into an AAV transgene cassette (see Materials and Methods), and AAV-flex-shRNA-GluN1 and AAV-flex-shRNA-scramble (scr)viruses were then bilaterally injected into the LPO areas of Vglut2-Cre and Vgat-Cre mice to generate Vglut2-shRNA-GluN1-LPO and Vgat-shRNA-GluN1-LPO mice and the associated scramble control respectively (Fig. 8C, Fig.   9A).

Sedatives and sleeping medications improve sleep in mice lacking NMDA receptors in LPO GABA neurons
Next, we tested the effects on sleep fragmentation in Vgat-shRNA-GluN1-LPO mice by Thus, sedatives and sleeping medications can transiently remove the insomnia (sleep-wake fragmentation) in mice lacking NMDA receptors in LPO GABA neurons.

Discussion
The PO hypothalamus is required for both NREM and REM sleep generation and NREM sleep homeostasis (Nauta, 1946;McGinty and Sterman, 1968;Sherin et al., 1996;John and Kumar, 1998;Lu et al., 2000;Lu et al., 2002;Szymusiak et al., 2007;Zhang et al., 2015;Ma et al., 2019;Reichert et al., 2019). We explored how NMDA receptors on LPO neurons regulate sleep. Deleting the core GluN1 subunit of NMDA receptors from LPO neurons substantially reduced the excitatory drive onto these cells and abolished activity during all vigilance states. These ΔGluN1-LPO mice had less NREM sleep and altered REM sleep patterns (atonia was present, but there was reduced EEG theta activation). In addition, ΔGluN1-LPO mice had highly fragmented sleep-wake: they had many more episodes of wake and NREM sleep, but each episode was shorter. Thus, although ΔGluN1-LPO mice can still enter NREM sleep from wake, AMPA glutamate receptor excitation alone on LPO sleep-promoting neurons is insufficient to maintain NREM sleep or produce REM sleep. The ΔGluN1-LPO mice phenotype is quite similar (wake-NREM fragmentation, loss of REM sleep) to mice with a double (global) deletion of the muscarinic receptor genes Chrm1 and Chrm3 (Niwa et al., 2018), so this could intersect on the same pathway. The phenotype was further stratified. High sleep-wake fragmentation, but not sleep loss, was produced by selective GluN1 knock-down in GABAergic LPO neurons (Vgat-shRNA-GluN1-LPO mice).
The molecular mechanism of sleep homeostasis, whereby the time spent awake is tracked and leads to an increase drive to sleep, is not resolved. A mutation in one kinase, saltinducible kinase3, which is expressed throughout the brain, reduces sleep homeostasis (Funato et al., 2016;Honda et al., 2018). In regions such as neocortex and hippocampus, increased time awake leads to increased phosphorylation of hundreds of synaptic proteins, including glutamate receptors (Wang et al., 2018;Bruning et al., 2019). Calcium entry through NMDA receptors has been suggested to be part of the sleep homeostasis mechanism that tracks time spent awake, and the calcium entry through NMDA receptors could stimulate phosphorylation (Liu et al., 2016;Tatsuki et al., 2016). But at least for the PO hypothalamus, which contains galanin neurons required for sleep homeostasis Reichert et al., 2019), our findings show that NMDA receptors are not needed for sleep homeostasis.
The sleep homeostasis process is reflected in changes in EEG delta power (Borbely et al., 2016). During the 24-hour cycle, delta power is highest during the "lights on" sleep phase and declines as each NREM sleep bout progresses (Fig. 6D, E), which is thought to reflect the dissipation of the homeostatic sleep drive (Borbely et al., 2016). After sleep deprivation, neocortical activity in the subsequent ("recovery") NREM sleep is deeper (more synchronized and thus has a higher delta power). However, we found that NMDA receptor deletion in LPO did not affect sleep homeostasis as defined by the classical criteria -EEG delta power showed its usual variation, an increase and decrease over 24 hours. Even placing ΔGluN1-LPO mice under high sleep pressure by sleep deprivation did not enable the mice to sleep well. The sleep fragmentation persisted even during the recovery sleep, and the fragmented sleep started with a higher delta power, as expected for recovery sleep in the sleep homeostasis model. So, the sleep homeostatic process seems independent of the mechanism maintaining consolidated sleep. In fact, during sleep deprivation, ΔGluN1-LPO mice made multiple attempted entries to sleep. It was as if the mice were chronically sleepy, but they nonetheless were not driven to sleep.
Many people suffer from occasional insomnia, but it can become a debilitating condition . Insomnia, as a clinical disorder, is defined as an inability to initiate or maintain sleep at least three times a week over three months, even when sleep conditions are otherwise optimal (Van Someren, 2020). Insomniacs frequently report that their sleep is nonrestorative and that they sleep less. In fact, insomnia sufferers often have the same amounts of EEG-defined NREM sleep as controls, but oscillate frequently between wake and NREM sleep, so that their sleep is fragmented (Van Someren, 2020). The Vgat-shRNA-GluN1-LPO mice, which have the same amount of sleep, but high sleep-wake fragmentation, could be a useful model for intractable insomnia. Miracca et al. Page 18 Behavioral therapy is often ineffective for treating intractable insomnia disorder, and medication remains an alternative approach if used cautiously (Shahid et al., 2012;An et al., 2020;Van Someren, 2020). Unlike sleep deprivation, which is usually efficient at inducing sleep, drugs could treat quite effectively the insomnia of ΔGluN1-LPO mice.
Dexmedetomidine could transiently restore consolidated NREM sleep. Dexmedetomidine, an α2 adrenergic agonist, induces stage 3 NREM sleep in humans and NREM-like sleep in animals (Gelegen et al., 2014;Zhang et al., 2015;Akeju et al., 2018), and requires galanin/GABA neurons in LPO for its effects . Zolpidem (Ambien), a GABA A receptor positive modulator, is a widely prescribed sleeping medication . Its main effect in humans is to reduce latency to NREM sleep rather than maintaining consolidated sleep. Nevertheless, zolpidem did have a beneficial effect on both ΔGluN1-LPO and Vgat-shRNA-GluN1-LPO mice, restoring longer periods of NREM sleep.
Our findings also demonstrate a new aspect of REM sleep generation. REM sleep is characterized by a high theta:delta frequency ratio in the EEG together with muscle atonia. In rodents, the theta itself detected in the cortical EEG seems to originate mostly from the hippocampus. Indeed, the theta activation during REM is required for memory processing (Boyce et al., 2016;Izawa et al., 2019). Although the brainstem circuitry that generates muscle atonia during REM sleep is reasonably well understood, the circuitry that produces the theta activity in the EEG during REM sleep is only partially characterized, seeming to require distributed circuitry throughout the forebrain (Renouard et al., 2015;Peever and Fuller, 2016;Luppi et al., 2017;Izawa et al., 2019;Yamada and Ueda, 2019), including the MCH NREM-REM-promoting neurons in the lateral hypothalamus (Jego et al., 2013), REM-off and REM-on neurons in the dorsal medial hypothalamus (Chen et al., 2018), and GABA and cholinergic neurons in the medial septum that project to the hippocampus (Yoder and Pang, 2005). Although LPO is known to be required for REM sleep (Lu et al., 2000;Lu et al., 2002), we were surprised to discover that LPO neurons, regardless of type (e.g. galanin, Vgat, Vglut2), have actually their highest activity during REM sleep. We found that GluN1 knockdown in GABA and Vglut2 cells of LPO did not influence REM sleep, whereas the pan knockout in all LPO neurons did, so the cell type(s) expressing NMDA receptors responsible for REM sleep generation in LPO require further investigation.
We speculate that NMDA receptor properties could be responsible for maintaining NREM and REM sleep promoting LPO neurons in the "on" state. In contrast to AMPA-gated ionotropic glutamate receptors, NMDA receptors stay open for around 100 msec to 1 s, as well as having a voltage-dependent magnesium block (Paoletti et al., 2013). Because of these properties, NMDA receptors have been intensely studied for their role in synaptic plasticity. But these same properties also allow NMDA receptors to act as pacemakers, controlling rhythmic firing e.g. in those circuits involved in breathing, swimming and walking (Steenland et al., 2008;Li et al., 2010), as well as contributing to the generation of burst firing of reticular thalamic neurons (Deleuze and Huguenard, 2016). The long open times of NMDA receptors, especially those located extrasynaptically, could be contributing to tonic excitation (Sah et al., 1989;Neupane et al., 2021), stabilizing hypothalamic sleep-on neurons in their firing mode. It will be interesting to see if this role of NMDA receptors generalizes to other sleep-promoting circuits. For example, we previously found that genetic silencing of mouse lateral habenula neurons with tetanus toxin light-chain produced high NREM sleep-wake fragmentation with conserved amounts of total sleep and wake (Gelegen et al., 2018). It seems likely that disrupting NMDA receptors on these cells would also produce insomnia, given that that NMDA receptors are needed to keep lateral habenula cells in burst firing (active) mode (Yang et al., 2018;Cui et al., 2019).
In conclusion, we have found that selectively reducing NMDA receptors in the LPO hypothalamic area causes insomnia (wake-NREM sleep fragmentation) and loss of theta activity during REM sleep. Given that sleep homeostasis is intact in mice with no NMDA receptors in LPO hypothalamus, the mechanism of sleep maintenance is distinct from that of the sleep drive itself.

Significance Statement
Insomnia is a common affliction. Most insomniacs feel that they do not get enough sleep, but in fact, often have good amounts of sleep. Their sleep, however, is fragmented, and sufferers wake up feeling unrefreshed. It is unknown how sleep is maintained once initiated. We find that in mice, NMDA-type glutamate receptors in the hypothalamus are the main drivers of excitation and are required for a range of sleep properties: they are, in fact, needed for both sustained NREM sleep periods, and REM sleep generation. When NMDA receptors are selectively reduced from inhibitory preoptic neurons, mice have normal total amounts of sleep but high sleep-wake fragmentation, providing a model for studying intractable insomnia. Data are mean ± SEM (*p < 0.05, **p < 0.005, ***p < 0.0005).    A, Grin1 lox mice were bilaterally co-injected in LPO with AAV-iCre-2A-mCherry and AAV-flex-GCaMP6s to record calcium levels from neurons lacking NMDA receptors.
An optic fiber was also implanted unilaterally for these recordings. On the right, immunohistochemistry showing from left to right: DAPI (blue) and AAV-flex-GCaMP6s transgene (green) on the first row, Cre recombinase (red) and merge on the second row. Scale bars represent 1 mm. B, example of a photometry recording aligned to EEG and EMG in a ΔGluN1-LPO mouse. From the top: vigilance state, delta power, EMG, EEG and    A, representation of 6 h SD protocol starting when lights turn ON (ZT0), using novel objects to keep the animals awake. For the remaining 18h animals are left undisturbed. B, Wake EEG power spectrum during 6h SD. C, number of sleep attempts during the 6h SD (left) and latency to fall asleep (right) after SD, considering the first NREM sleep bout as at least 30s long. D and E, left panels, NREM sleep EEG power spectrum during 1h following 6 h SD compared with same circadian time during baseline recordings in ΔGluN1-LPO (D) and in GFP-LPO (E) mice; right panels, NREM sleep EEG delta power calculated for every hour during baseline recordings and after 6h SD in ΔGluN1-LPO and GFP-LPO animals. The EEG power was normalized over the total power during each hour. F, percentage of Wake (left) and NREM sleep amounts (right) in ΔGluN1-LPO and GFP-LPO mice over their own baseline after 6 h SD. ZT6-12 (L2), ZT12-18 (D1), ZT18-24 (D2). G, sum of state transitions in the 18h following 6h SD presented as a percentage over baseline for both ΔGluN1-LPO and GFP-LPO mice during the same circadian time. H, left panel, episode number calculated over every 6 h following SD for Wake (left), NREM (center) and REM sleep (right). Right panels, episode mean duration calculated by 6 h following SD for Wake (left), NREM (center) and REM sleep (right). GFP-LPO, n=7; ΔGluN1-LPO, n=7. Data in all panels B, C, D, E, F and H are represented as means ± SEM (*p < 0.05, ** p < 0.005, *** p < 0.0005, † p < 0.00005). difference from baseline values for each drug and dose tested. Asterixis in red, green, and blue indicated significant differences from BL values only for Dex 25μg/kg, Dex 50 μg/kg, and zolpidem respectively.